1.1 Field of the Invention
The invention relates to devices for performing chemical reactions, and in particular automated devices for performing polymerase chain reaction (PCR). Still more particularly, the present invention provides methods and apparatus for controlling reaction temperatures in an automated PCR device. The present invention has applications in the areas of analytical chemistry, molecular biology, and process chemistry.
1.2 The Related Art
Polymerase Chain Reaction (PCR) has become a mainstay of biochemical laboratories, and with good reason: this elegant method of replicating oligonucleotides using polymerase enzymes, such as Taq polymerase, has been a major factor in the explosion of biotechnological research and products. The process and its applications are well known those persons having ordinary skill in the art (Ausubel, Brent et al. 1992). In brief, PCR enables the rapid replication of oligonucleotides, in particular DNA, so that single copies of an oligonucleotide can be transformed into a significant concentration to enable further manipulation or analysis. The PCR procedure involves: transferring target oligonucleotide from a sample into a crude extract; adding an aqueous solution containing various enzymes, buffers, triphosphates (dNTPS), and complimentary oligonucleotide primers to the extract to form a reaction mixture; cycling the temperature of the reaction mixture between two or three temperatures (e.g., 90° C.-96° C., 37° C.- 65° C., and 72° C.) repeatedly to enable replication of the target oligonucleotides; and then detecting the amplified oligonucleotides. Intermediate steps, e.g., purification of reaction products and incorporation of surface-bending primers, also may be included. Each cycle doubles the number of target oligomer sequences. PCR techniques can amplify a single molecule of an oligonucleotide or oligoribonucleotide about 106-to about 109-fold.
Because genetic information can be used to establish the identities of individuals and the types of organisms, and because PCR is capable of creating analyzable quantities of genetic material from very small samples, many technologies exist to facilitate the application of PCR to analytical and forensic tasks. In particular, the use of PCR to identify unidentified bodies and criminals is widely used in law enforcement and the military. The application of PCR to national defense and homeland security is also an area of active biodefense development. Also, doctors and hospitals increasingly want access to portable PCR technology for use in care giving. Examples of such uses include:                Screening blood, saliva, or urine samples for multiple infectious diseases associated with upper respiratory, intestinal, or STD infections.        Determining if an infectious disease is resistant to antibiotics.        Determining if an infection is viral or bacterial.        Identifying an individual's susceptibility to an adverse drug reaction.        Diagnosing a cancer type (e.g., breast, prostate, ovarian, pancreatic).        Identifying an individual's predisposition to Alzheimer's disease.        
These applications have created a heavy demand for automated PCR apparatuses that can process small sample concentrations on-site at remote locations to provide analytical data to investigators and medical workers.
As note above, a fundamental operation during the PCR process is thermal cycling, i.e., the raising and lowering of reaction temperatures to enable the amplication process, in which the temperature of the reaction mixture is driven between about 60° C. and about 95° C. as often as fifty or more times. A thermal cycle typically has four segments: heating the sample to a first temperature; maintaining the sample at the first temperature; cooling the sample to a lower temperature; and maintaining the temperature at the lower temperature. Conventional PCR instrumentation typically uses an aluminum block holding as many as ninety-six conical reaction tubes in which the sample and necessary reagents for amplication are contained. The block is heated and cooled during the PCR amplication process, often using either a Peltier heating/cooling apparatus, or a closed-loop liquid heating/cooling system in which flowing through channels machined into the aluminum block. However, the large mass of the aluminum block, and the conductivity of aluminum, limit the rates of heating and cooling to about 1° C. per second; so a fifty-cycle PCR amplification process takes at least about two hours.
Moreover, the cooling rate of the aluminum block is significantly lower than the heating rate. The asymmetry between the heating and cooling rates reduces the efficiency of the PCR process. For example, unwanted side reactions can occur at temperatures between the extremes creating unwanted DNA products, such as so-called “primer-dimers” and anomalous amplicons that consume reagents necessary for the desired PCR reaction. Other processes, e.g., ligand binding (organic or enzymatic) also suffer from unwanted side reactions under non-uniform temperatures that often degrade the analysis. For these reasons, optimization of the PCR process and similar biochemical reaction processes requires that the desired optimal reaction temperatures be reached as quickly as possible, spending minimal time at intermediate temperatures. Therefore the reaction vessels containing the reactants must be designed to optimize heating and cooling rates, to permit real time optical interrogation, and to accept various sample volumes.
One automated PCR system is the MATCI device disclosed in U.S. Pat. No. 5,589,136 (Northrup, Raymond P. Mariella et al. 1996), which describes a device that uses a modular approach to thermal cycling and analysis: Each reaction is performed in its own thermal cycling sleeve, and each sleeve has its own associated optical excitation source and fluorescence detector. The low thermal mass of the thermal cycling sleeve allows the MATCI device to realize extremely fast thermal cycling: samples can be heated at a rate of up to 30° C./sec. and cooled at rate as great as 5° C./sec. Two other commercially available systems, sold under the trade names GeneXpert (Cepheid, Sunnyvale, Calif.) and Razor (Idaho Technology, Inc.), use disposable fluidic cartridges, each containing an flexible reaction chamber that expands under pressure to make tight contact with a solid heater located in the instrument (Petersen, McMillan et al. 1999). The Razor uses a flexible fluidic pouch and actuators that move a reaction slug within the pouch; the reaction zone walls of the pouch make tight contact with two solid heaters. In both cases, the heater is a solid and the disposable cartridge or pouch contains one or more reaction zones, each with a thin, flexible wall that makes thermal contact with the heater. Still another technology, sold commercially under the trade names TruDiagnosis™ and TruArray™ by Akonni Biosystems (Fredericksburg, Md.), rapidly screen a sample for hundreds of disease markers at one time by using hundreds of molecular biosensors arrayed in a microarray the size of a fingernail. The samples are conveyed through the array using microfluidic channels. The Akonni technology can provide accurate diagnostic results in less than 30 minutes to support an informed and timely treatment decision.
Nevertheless current approaches to handling thermal cycling are limited, depending on flexibility in the disposable component to create satisfactory thermal contact with the instrument hardware, are needed. In particular, methods and apparatus that provide the desired cycling performance without reliance on special reaction chamber materials offer the promise of reduced cost and greater efficiency. The present invention meets these and other needs.